Proteins play a variety of key roles in biological processes and functions, including for example, functioning as catalysts, regulators of biochemical pathways, receptors, and as important elements in immune response. Given their diverse and important roles, it is not surprising that ligands that bind to proteins have been viewed by pharmaceutical researchers as attractive candidates for therapeutic agents. One traditional approach for drug discovery simply involved making modifications to natural regulators. As more data regarding structure function relationships became available, it became possible to engage in rational drug design using computers and x-ray structures to aid in synthesizing molecules tailored to fit the active site of an enzyme, for example. However, even using such advanced techniques, drug screening and development remained an often tedious and time consuming process.
More recent drug discovery methods take a different approach and involve screening extremely large libraries of compounds for their ability to bind protein targets of interest. This type of approach typically begins with the identification of a potential protein target, such as a receptor for example. A diverse library is then prepared containing ligands to be screened for their ability to bind the target. The libraries may be random peptide libraries, carbohydrate libraries, natural product libraries, etc. Often the libraries are prepared using recently developed combinatorial techniques. These libraries are subsequently subjected to high throughput screening to identify ligands that bind to the target. Because the key feature of this approach is to screen a huge number of molecules, the success of this approach hinges on the ability to rapidly screen and identify ligands that do bind the target. Ligands initially identified as binding the target are then used to develop more focused libraries that are then put through the same screening process. This process of screening and preparing new focused libraries typically is repeated several times until a relatively small population of lead compounds are identified. These lead compounds are then subjected to various pharmaceutical analyses to select useful drug candidates.
A primary limitation in current methods is that the screening tests simply detect binding, but are unable to distinguish between specific and non-specific binding. Some approaches also are not fully compatible with high-throughput screening procedures. Moreover, many current screening methods require labeling of either the target or ligand and are unable to detect binding complexes directly.
The present invention provides novel methods for analyzing protein binding events in which the formation of protein/ligand complexes can be directly detected. Using this system, it is possible to screen libraries on the basis of specific binding interactions. It is also possible to perform a variety of analytical and diagnostic analyses with the system of the present invention.